Oligonucleotides that are complementary or "antisense" to specific genes or RNA sequences are relatively small, synthetic molecules having an average molecular weight of about 10 kilodaltons (kD). These antisense molecules have had widespread use in the field of selective gene regulation with consequent therapeutic implications. Phosphate backbone modification of such oligonucleotides provides nuclease resistance and greatly enhances the usefulness of these analogs. Such modifications include the substitution of phosphodiester internucleotide linkages with linkages such as methylphosphonates (Murakami et al. (1986) Biochem. 24:4041-4046; Agrawal et al. (1987) Tetrahedron Lett. 28:3539-3542; Sarin et al. (1988) Proc. Nat. Acad. Sci. (USA) 85:7448-7451), phosphorothioates (Burgers et al. Biochemistry 18:592-596; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Agrawal et al. (1989) Nucleosides and Nucleotides 8:819-823; Agrawal et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:7790-7794), and phosphoramidates (Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Agrawal et al. (1989) Nucleosides and Nucleotides 8:819-823).
Of special interest are phosphorothioate analogs in which one non-bridging oxygen atom has been substituted for a sulfur atom on the phosphate group in each internucleotide phosphate linkage. This modification is a conservative substitution which increases nuclease resistance without significantly impairing the hybridization of the antisense molecule with target mRNA. As synthesized, these modified oligonucleotides or analogs are usually found as diastereomeric mixtures due to chirality at their phosphorous group. In a context of new drug research, development and manufacturing of such analogs requires that the issues of oligomer length, base composition, base sequence, chemical purity, and stereochemical purity be successfully addressed.
Synthetic oligonucleotides are presently used in most laboratories using molecular biology techniques. As synthesized, these oligonucleotides generally exist as mixtures of truncated oligonucleotides in addition to the desired oligonucleotide. Since the purity and chemical identity of a particular oligonucleotide is crucial to many applications, the ability to characterize and separate synthetic oligonucleotides analogs on a routine basis is important.
The absolute length and the degree of length heterogeneity of prepared oligonucleotides have been assessed by electrophoresis in high resolution denaturing polyacrylamide slab gels (PAGE) (see e.g., Current Protocol in Molecular Biology, Green Publishing and Wiley Interscience, New York, 1988) and by capillary gel electrophoresis through cross-linked polyacrylamide (6% T, 5% C) gels (Hjerten (1967) Chromatogr. Rev. 9:122-213) containing from 10% to less than 30% (vol.:vol.) formamide (Rocheleau et al. (1992) Electrophoresis 13:484-486). Detection of oligonucleotides separated on such gels has been accomplished by autoradiography and laser-induced fluorescence. These methods have not proven suitable for separating modified oligonucleotides. Furthermore, some of these gels, once used, are not easily removable from the capillary. To remedy this problem, gels containing up to 5% acrylamide monomer have been polymerized before filling the capillary (EPO 497 480). Ultrathin slab gels (less than 100 .mu.m in thickness) have also been used for high speed DNA sequencing (Brumley et al. (1991) Nucleic Acids Res. 19:4121-4126; Ansorge et al. (1990) Nucleic Acids Res. 18:3419-5420). Alternative separation methods include ion exchange chromatography, reversed phase high pressure liquid chromatography (HPLC), and gel high performance capillary electrophoresis (HPLC) (see e.g., Edge et al. (1981) Nature 292:756-762; U.S. Pat. No. 4,865,707).
Oligonucleotides with phosphorothioate linkages are more difficult to resolve than phosphodiester-linked DNA due to the existence of diastereomer isomers (2.sup.n, where n=the number of chiral centers, which is equivalent to the number of phosphate groups). In addition, difficulty in resolution may be due to increased hydrophobicity of the former. These molecules, when separated, interact hydrophobically with ion exchange column supports and in many cases co-elute. Thus, they cannot be separated by the above methods in their existing formats.
The separation of phosphorothioate oligonucleotide analogs is problematic for other reasons as well. When phosphorothioate oligonucleotides are assembled using either methoxyphosphoramidite or H-phosphate chemistry, they are in the form of diastereomeric mixtures due to chirality at their phosphorous groups. As a result, although they migrate through polyacrylamide gels and HPLC columns like their corresponding phosphodiester counterparts, phosphorothioate oligonucleotides give broader peaks and run more slowly than phosphodiesters because of their increased hydrophobicity. They are also known to interact with the HPLC column support. In addition, phosphorothioates run into stereochemical problems when separated by reversed phase HPLC. General analytical methods have not been devised for establishing the ratio of the optical isomers at each unsymmetrical substitution phosphorous linkage in an analog having many such sites of local chirality.
HPLC of oligodeoxyribonucleotides containing one or two phosphorothioate internucleotide linkages using a reversed-phase column (RP-HPLC) has been reported (Stec et al. (1985) J. Chromatogr. 326:263-280; Agrawal et al. (1990) Nucleic Acids Res. 18:5419-5423). However, this method is of limited use because of the small differences in the hydrophobicity of these analogs with increasing chain length (Agrawal et al. (1990) J. Chromatogr. 509:396-399).
Separation of oligodeoxyribonucleotide phosphorothioates containing 10 or fewer nucleotides has also been achieved by HPLC on strong anion-exchange (SAX) columns (Agrawal et al. (1990) J. Chromatogr. 509:396-399). In this method, oligonucleotide phosphorothioates were converted to their phosphodiester counterparts in one step, and then were analyzed by HPLC. Unfortunately, oligonucleotides phosphorothioates containing more than 10 nucleotides can not be analyzed by this method because of their strong interaction with the SAX medium. Thus the separation of oligonucleotide phosphorothioates by this method is limited by its oligonucleotide length dependency.
Length-dependent separation of phosphorothioate analogs by HPLC using a weak anion-exchange (WAX) column has also been accomplished (Meletev et al. (1992) Analyt. Biochem. 200:342-346). However, the peaks obtained were broader than those obtained for their phosphodiester counterparts, possibly because of their diastereomeric backbone. Ion-pair HPLC has also been used to analyze oligonucleotide phosphorothioates (Bigelow et al. (1990) J. Chromatogr. 533:131-140), but length-dependent separation was not achieved.
Thus, what is needed are better analytical methods of separating unmodified and modified mononucleotides and oligonucleotides cleanly, rapidly, efficiently, and which are not limited by the size range or modification of the molecules being analyzed. In addition, methods enabling the quick and easy removal and replacement of the substrate used for separation are also needed.